于彥麗, 李艷嬌, 龐凱元, 張發(fā)軍, 孫琦, 李文才, 孟昭東

山東省農業(yè)科學院玉米研究所, 小麥玉米國家工程實驗室, 濟南 250100

植物FKBP基因家族的結構及生物學功能

于彥麗, 李艷嬌, 龐凱元, 張發(fā)軍, 孫琦, 李文才, 孟昭東

山東省農業(yè)科學院玉米研究所, 小麥玉米國家工程實驗室, 濟南 250100

FK506結合蛋白(FK506 binding protein, FKBP)是一種在生物體中廣泛存在、進化上高度保守的組成型蛋白質。除了作為免疫抑制劑FK506受體以外, FKBP還具有肽脯氨酰順反異構酶(PPIase)的活性。FKBP在植物中是個大家族, 可作為分子伴侶與一些蛋白相互作用從而調控不同的生化過程。研究表明, 該家族基因在植物的響應脅迫和不同生長發(fā)育過程中都扮演著重要角色。最近許多新的FKBP互作蛋白的發(fā)現和鑒定表明, FKBP在調控基因表達和光合適應性方面具有廣泛的生物學功能。文章對植物FKBP家族的結構特點、分類以及最新的功能研究進展進行了詳細的綜述。

FKBP; 結構特點; 生物學功能; 親免素

親免素(Immunophilin)是近年來發(fā)現的可與免疫抑制劑環(huán)孢霉素A(Cyclosporine-A, CsA)、FK506及雷帕霉素結合的一類細胞受體蛋白, 廣泛地存在于生物體內, 且含量豐富[1]。根據結合的免疫抑制劑不同, 親免素家族分為兩類：與環(huán)孢素 A結合的蛋白稱為親環(huán)素(Cyclophilin, CyP); 另一類可與FK506和雷帕霉素特異結合, 稱為FKBP[2,3]。自20世紀80年代初期發(fā)現親免素以來, 人們已經成功分離純化出多種 FKBP, 并且發(fā)現其與免疫抑制劑之間的相互作用, 預示著親免素在器官移植領域有著巨大的潛力。免疫抑制劑已經被廣泛地應用于自身免疫疾病和器官移植等多個方面, 親免素可以與免疫抑制劑結合形成親免素-免疫抑制劑復合物(FKBP-FK506, CyP-CsA), 在一定程度上可有效緩解機體內的排異反應[4]。另外, FKBP與正常的神經系統(tǒng)生化活動具有十分密切的聯(lián)系, 可作為特異性小分子配體, 在治療神經退行性疾病和促進神經再生等方面有著潛在的研究價值和臨床應用價值[5]。在免疫細胞中, 親免素-免疫抑制劑復合物可與鈣調磷酸酶(Calcineurin, CaN)相互作用, 從而阻遏免疫相關基因表達過程中的磷酸化信號途徑, 產生免疫抑制作用; FKBP-雷帕霉素復合體在細胞內的靶蛋白不是 CaN, 而是雷帕霉素靶體 TOP(Target of rapamycin), 通過與之結合為三元復合體, 抑制細胞周期的正常進行, 因此發(fā)揮免疫抑制的作用[6]。親免素的一個主要特征是具有肽脯氨酰順反異構酶(Peptidyl-prolyl cis-trans isomerase, PPIase)活性, 即催化多肽或蛋白質底物中脯氨酸殘基N端肽鍵的構象由順式轉為反式[7～9]。肽鏈末端肽鍵的順式反式異構體之間的自由轉換對于新生蛋白和生物大分子來說, 是其正確折疊和正確自我組裝所必需的[7～9]。除了具有PPIase酶活性, FKBPs還充當著分子伴侶的角色, 提供蛋白質之間相互作用的結合位點[10]。

自1989年首次發(fā)現FKBP[7,8]以來, 關于該基因家族的研究大部分集中在酵母和哺乳動物中, 而在高等植物中的研究相對較少。FKBP基因家族在擬南芥(Arabidopsis thaliana)[11]、水稻(Oryza sative L.)[12]和玉米(Zea mays L.)[13]中分別有23、29和24個成員。對植物FKBP基因家族研究顯示, FKBP參與細胞信號轉導、蛋白定位、蛋白折疊和轉運等過程, 對植物的生長發(fā)育具有一定的調節(jié)作用, 并且可以調控激素信號的轉導, 在植物抗逆生理中起重要作用[14～16]。本文針對植物 FKBP家族, 對其結構、進化與分類以及各種生物學功能等方面的研究進行了綜述, 以期為科研工作者對其基因新功能的挖掘以及基因功能的應用奠定基礎。

1 植物FKBP的結構域組成

1.1 FK506結合域

FKBP蛋白家族的每個成員都至少含有一個FK506結合域(FKBd), 該結構域既是FK506和雷帕霉素的結合位點, 也是 PPIase催化作用的活性位點。FKBd大約含有110個保守的氨基酸序列, 且三級結構高度保守[17～19]。FKBd由6條反向平行的β鏈構成的β折疊、一段短的α螺旋以及30s、40s、50s、80s四段柔性連接環(huán)區(qū)(根據其在FKBP序列上的位置來命名)組成(圖 1)。β折疊可形成一個正對α螺旋的凹面, 核心蛋白形成疏水腔可結合脯氨酸, 疏水側鏈伸向該疏水腔[20～22]。盡管FKBd在結構上嚴格保守, 還是有相當一部分的 FKBP表現出低或無PPIase活性(表1), 說明該結構域可能有其他的功能。

1.2 多結構域FKBP

圖1 FKBd結構模型H. sapiens FKBP12的三級結構

表1 植物中FKBP家族已知成員

(續(xù)表1)

植物FKBP家族成員的大小變化范圍非常廣泛,如擬南芥AtFKBP12是只有一個FKBd組成的單結構域(Single-domain, SD)的FKBP, 其分子量只有12 kDa,而小麥 wFKBP77是具有多個結構域(Multidomain, MD)的FKBP, 其分子量為77 kDa[43]。植物及其他物種中的大分子量的 FKBP除了含有基本的結構域FKBd外, 還含有其他結構域：TPR(Tetratricopeptide repeat, 三十四肽重復序列)形成反向平行的α螺旋結構域, 可與熱激蛋白 HSP90結合[42]; CaMBds (Calmodulin-binding domains, 鈣調素結合結構域)通常位于植物大分子量 FKBP的 C端并且能夠與CaM(Calmodulin, 鈣調素)結合[28,38](圖 2)。盡管植物FKBP在鈣離子信號途徑中的作用目前還不清楚,但是哺乳動物 FKBP38的 TPR區(qū)可以和熱激蛋白HSP90結合, 末端FKBd與靶蛋白結合, C端CaMBd可以受到Ca離子信號調控, 從而激活其PPIase和分子伴侶的活性[58], 這預示著多結構域的植物 FKBP可能也具有類似的鈣調控機制?？傊? 對于多結構域 FKBP來說, 其復雜的結構也將賦予其更加復雜和重要的功能。

圖2 FKBP結構示意圖AtFKBP12代表單結構域 FKBP, wFKBP77代表多結構域 FKBP。藍色方框代表 FKBd(FK506結合域), 紅色方框代表TPR(tetratricopeptide repeat, 三十四肽重復序列), 黃色方框代表 CaMBds(鈣調素結合結構域), 棕色方框代表細胞核定位信號(nuclear localization signals), 數字表示氨基酸位置。

2 植物FKBPs的生物學功能

2.1 FKBP家族成員的抗逆功能

擬南芥 AtFKBP62和 AtFKBP65通常被稱作ROF1和ROF2, 是多結構域的FKBP, 它們之間的同源性達到 85%, 二者是通過基因復制產生, 互為旁系同源物。Breiman等[39,40]研究發(fā)現ROF1和ROF2通過調控高溫脅迫修復相關的小熱激蛋白(sHSPs)的表達來響應高溫脅迫, 但是二者在擬南芥長期遭受高溫時的發(fā)育過程中卻具有相反的作用。ROF1通過其TPR與HSP90結合, HSP90又與熱激轉錄因子HsfA2結合, ROF1-HSP90-HsfA2復合體轉運到細胞核內誘導sHSPs和ROF2的表達[39]。ROF2通過與 ROF1的 FKBd結合, 破壞核內 ROF1-HSP90-HsfA2復合物, 因而可以負調控 sHSP的表達[40]。rof1基因敲除后, 植物熱激再恢復2～3 d后幾乎完全喪失抵抗高溫的能力, HsfA2基因敲除后表型與rof1相似, 但是rof2突變體和ROF1超表達植株與野生型對照相比, 獲得了更強的持續(xù)耐熱的能力[39,40]。ROF2最近被證實通過控制K+內流來調控細胞內pH值和細胞膜的極性, rof1/rof2雙突變體比rof1或rof2任一單突變體對于酸抑制都更加敏感, 說明 ROF1和ROF2功能協(xié)同[41]。

在小麥[35,43]、水稻[36]和玉米[13]中都存在 ROF1/ ROF2的同源蛋白, 它們的結構及表達模式都與擬南芥同源基因相似, 說明在高等植物中, 這對旁系同源FKBP在獲得性耐熱機制中的拮抗作用也是保守的。哺乳動物中FKBP52/FKBP51是ROF1/ROF2的同源蛋白, 它們通過TPR與HSP90結合, 進而結合到糖皮質激素或其他一些類固醇受體上響應脅迫[59]。同ROF1和ROF2一樣, FKBP52和FKBP51在脅迫響應中的作用也相反。FKBP52協(xié)助與之結合的糖皮質激素進入細胞核, 促進包括 FKBP51在內的脅迫響應基因的表達, FKBP51抑制糖皮質激素的轉運[50,60]。類似于ROF1/ROF2的旁系同源物在脅迫響應過程中存在微調機制, 由此推測其他 FKBP旁系同源物可能也存在相似的調控方式。

水稻OsFKBP20-1a和OsFKBP20-1b具有85%的同源性, 盡管組織表達模式不同, 但是都受高溫和干旱誘導。Ahn等[61]報道, OsFKBP20-1a在所有植物組織中都有高豐度表達, 在高溫和干旱脅迫下表達量迅速上升, 然而OsFKBP20-1b只在脅迫處理24 h后表達量才開始升高。二者都定位于細胞核中, 而OsFKBP20-1b 同時還存在 于細胞質中[61]。OsFKBP20-1a與SUMO結合酶(Sce)結合, Sce又可通過小類泛素修飾蛋白(Small ubiquitin-like modifier, SUMO)與其他靶蛋白結合, 從而參與高溫脅迫響應[49], 但是目前還未發(fā)現與OsFKBP20-1a互作的蛋白。擬南芥中參與脅迫響應的同源復制FKBP是定位于內質網上的AtFKBP15-1和AtFKBP15-2, 二者同源性達到 70%且都受高溫誘導, 但具體功能目前還不清楚[11,48]。我們在玉米中也發(fā)現了旁系同源的FKBP, ZmFKBP15-3明顯受高溫誘導, 而 ZmFKBP15-1和ZmFKBP15-2在高溫條件下表達量與正常水平一致[13]。

2.2 FKBP家族成員在植物生長發(fā)育過程中的作用

由于擬南芥atfkbp42突變體發(fā)育遲緩而且根莖呈螺旋生長, 因此 AtFKBP42也被稱作 TWD1(TWISTED DWARF1), 這種表型是由于TWD1與胞內生長素外流相關蛋白 ABC(ATP-binding cassette)的結合影響了生長素運輸而導致[29,33]。進一步研究表明, TWD1通過其單一的FKBd結合到ABCB1(P-糖蛋白PGP1)和ABCB19(P-糖蛋白PGP19)的C端,調控質膜上生長素轉運系統(tǒng)的形成[29,30,33,62,63]。目前已從質膜和液泡膜上分離得到 TWD1[28], 該蛋白是通過其C端的膜錨定肽結合到膜上[31,64]。近來有研究表明TWD1位于內質網的胞質面, 并在該位置行使ABCB分子伴侶的功能從而進入分泌途徑[32], 目前這一結論還存有爭議。ABC家族成員ABCC1和ABCC2也稱為多藥耐藥蛋白 MRP1和 MRP2[33,65],可以參與液泡內砷、鎘、汞的轉運, ABCC1和ABCC2中恰好具有可與 TWD1 的 TPR結合的 CaMBd[66],但是TPR-CaMBd結合并不受體內Ca2+和CaM的影響[33], 并且其對于鈣信號轉導是否具有調控作用,仍有待于進一步驗證。TWD1的TPR結構域也可以和HSP90結合[28], 但是該結構域的作用仍不明確。TWD1的C端具有一個CaMBd, 該結構域對TWD1與膜轉運蛋白ABCC1和ABCC2的結合沒有任何影響, 其具體功能仍然未知。

超長鏈脂肪酸(Very long chain fatty acids, VLCFAs)是許多脂類的重要組成成分, 如膜上的磷脂和鞘脂, 它們是細胞正常分裂和分化所必需的。FKBP72由于參與VLCFAs的合成, 因而在植物發(fā)育中起重要作用[67]。FKBP72功能被破壞后使VLCFAs合成中斷, 進而導致細胞分裂發(fā)生紊亂[47], 產生擬南芥 atfkbp72突變體表型“PASTICCINO”[24,44]。AtFKBP72(PAS1)可以和參與VLCFAs合成的酶結合,如脫水酶(PAS2)、碳酸酶(PAS3)和還原酶(CER10, ECR)等, 因此被稱為內質網上組裝VLCFAs延伸復合體的腳手架[47,68,69]。PAS1也可以作為分子伴侶協(xié)助FAN(FKBP-associated NAC)轉錄因子轉運到細胞核中, 調控細胞的增殖[46]。PAN1均是通過其C端的CaMBd與VLCFA延伸復合體或FAN進行結合[45],預示這種結合可能受到CaM的調控。與TWD1類似, PAS1的 C端緊鄰 CaMBd處有一個膜定位信號肽,使得 PAS1一直被定位在質膜上, 因而在細胞分裂過程中, 不能進入細胞核來調控細胞分化[46]。直到與CaM或FAN結合后, PAS1接受細胞分化的信號,才能夠從質膜上分離, 由其 N端的核定位信號引導復合體進入到細胞核中[12]。

FKBP12是植物中最小的FKBP, 定位于細胞質中[13,16]。擬南芥AtFKBP12可與AtFIP37結合, At-FIP37是DNA結合蛋白, 參與mRNA剪接、細胞周期調控和胚胎發(fā)育[24,27]。在青杄中, PwFKBP12可與HAP類轉錄因子的亞基HAP5結合, 調控花粉管極性生長[26]。這些都說明植物FKBP12可以調控DNA結合蛋白的核運輸。

2.3 葉綠體FKBP在植物光合反應系統(tǒng)中的調控作用

2.3.1 調控光合膜的組裝

蛋白質組學分析表明在擬南芥中有相當多的FKBP與類囊體有關[70,71]。通過對高等植物 FKBP基因信號肽進行分析, 預測有30%～50%的FKBP定位于類囊體[11～13]。擬南芥類囊體PPIase活性檢測表明, 在類囊體中僅有FKBP13和親環(huán)素CYP20-2具有PPIase活性[55,72], 然而二者在葉綠體中功能冗余,所以親免素的PPIase活性在葉綠體中是否具有重要作用存在爭議[73]。近期一些研究表明, 位于葉綠體腔內的多數FKBP的主要功能是調控光合膜的組裝。PSⅡ(光系統(tǒng)Ⅱ)對植物生長至關重要, 擬南芥FKBP20-2功能缺失后, 導致 PSⅡ的蛋白超級復合物含量下降, 而未組裝的PSⅡ的單體和二聚體含量上升, 說明AtFKBP20-2 對PSⅡ的穩(wěn)定性和PSⅡ的組裝具有一定的影響[57]。Gollan等[54]通過酵母雙雜交實驗證明小麥TaFKBP16-1與PSⅠ(光系統(tǒng)Ⅰ)的亞基PsaL之間存在相互作用, 說明TaFKBP16-1參與 PSⅠ的形成, 而 TaFKBP16-3被證實可與 Thf1 (Thylakoid formation-1)和 APO2(Accumulation of PSI-2)結合, 后兩者均參與光合膜形成。此外, 該課題組還發(fā)現 TaFKBP13可與光合電子傳遞的主要載體 Cyt b6f (類囊體細胞色素 b6f復合體)的亞基Rieske鐵硫蛋白結合。Gupta等[53]早期通過酵母雙雜交實驗發(fā)現Rieske蛋白與AtFKBP13的信號肽特異結合, 并推測AtFKBP13是Rieske蛋白組裝的調控因子。但是在atfkbp13功能缺失突變體中, Rieske蛋白并沒有積累[73], 而且Rieske與類囊體腔內成熟的 TaFKBP13互作, 并不與其葉綠體中的前體形式結合[54], 因此 FKBP13-Rieske相互作用的意義仍有待進一步驗證。

2.3.2 調控光合作用過程中的電子流動

最近研究發(fā)現 AtFKBP16-2位于類囊體蛋白NDH(NADPH脫氫酶復合體)中, 并且在NDH的組裝及 NDH-PSⅠ超級蛋白復合體的形成等方面具有重要作用[75]。植物FKBP13和FKBP16-2是旁系同源蛋白, 它們都在相同的位置具有半胱氨酸對[11,12], AtFKBP13的半胱氨酸對經過氧化可形成二硫鍵,從而使活性位點更加穩(wěn)定并可結合靶蛋白[56,75], 因此有可能 FKBP13結合靶蛋白的活性, 是由類囊體內的氧化還原信號所調控。由此類推, FKBP16-2也具有結合靶蛋白的活性且受類囊體內氧化還原信號所調控。作為環(huán)式電子傳遞的載體, NDH和Cyt b6f的組裝及活性都是經由這些FKBP與氧化還原作用聯(lián)系起來[74,76,77]。C4植物的光合效率明顯高于 C3植物歸功于它的維管束鞘細胞, C4植物維管束鞘細胞中FKBP13和FKBP16-2含量顯著高于葉肉細胞[78],也間接說明FKBP在光合電子傳遞過程中發(fā)揮一定的作用。

3 植物FKBP調控基因的表達

FKBP除了可以參與調控植物生長發(fā)育和脅迫響應外, 最近有報道證明擬南芥 FKBP53參與調控核糖體基因的表達[51]。AtFKBP53是定位于細胞核中的多結構域 FKBP, 通過其 N端的酸性氨基酸結構域與核小體中的組蛋白 H3結合, 從而在染色質水平上抑制核糖體RNA基因(18S rDNA)的表達, 這表明AtFKBP53參與核小體組裝及基因的轉錄調控。在AtFKBP53的旁系同源蛋白 AtFKBP43的N端同樣存在酸性氨基酸結構域, 說明二者可能具有相同功能, 即功能冗余, 所以擬南芥突變體 atfkbp43和atfkbp53均沒有明顯表型[51]。

4 植物FKBP家族的進化

植物在進化過程中普遍存在基因復制現象, 所謂基因復制是指基因通過不等交換、逆轉錄轉座或全基因組重復等途徑產生一個與原基因相似的基因或堿基序列[79]。植物FKBP家族中有部分成員通過基因復制產生旁系同源物, 如擬南芥中AtFKBP15-1與AtFKBP15-2、AtFKBP62與AtFKBP65互為旁系同源物[11]。大多數高等植物FKBP沒有旁系同源物,如TWD1、PAS1和FKBP12, 它們通常都具有PPIase活性且在植物發(fā)育過程中擔任重要角色。與通過基因復制產生的FKBP相比, 它們與非植物FKBP間具有更遠的進化關系。說明該類FKBP具有較快的進化速率, 促使其典型的FKBd出現變異, 因而作為分子伴侶可以和更多的蛋白結合。另一方面, 在某些植物中, 有近一半數量的 FKBP是基因復制的產物, 互為旁系同源物的 FKBP可以結合相同的靶蛋白。當經過一系列進化, 它們的FKBd的loop區(qū)產生變異, 各自又可以結合不同的底物。Riggs等[80]報道, 哺乳動物FKBP51原本抑制激素受體的轉運,僅僅使其 80s loop上的一個氨基酸發(fā)生突變, 它就能像其旁系同源物FKBP52一樣促進激素受體的轉運。在很多旁系同源物中存在這種功能相反的現象,如之前所述的ROF1和ROF2, 為植物響應脅迫提供了微調機制, 也預示其他類似旁系同源物如FKBP15-1和 FKBP15-2、FKBP43和 FKBP53及FKBP13和FKBP16-2間具有類似功能。

高等植物FKBP多基因家族的進化是通過外顯子跳躍和基因復制[12,18,81], 最初的 FKBP通過其疏水腔的脯氨酸基序與靶蛋白結合, 隨著復制基因關鍵位點突變的產生, FKBP的靶蛋白逐漸出現多樣性。進化的另一個結果就是在多結構域FKBP中, 如TWD1、ROF1/ROF2, 除了FKBd外還出現了TPR、CaMBds以及膜定位信號等結構域, 使得這些FKBP可與特定靶蛋白、其他蛋白復合體、轉運蛋白及膜結合, 從而行使更多的功能。FKBP被稱為調控某些蛋白活性的分子開關, 一種可能是因為其具有的PPIase活性可以調控脯氨酸的構象[82], 另一種可能是 FKBP可以結合到特定的脯氨酸基序上, 從而調控靶蛋白和其他蛋白或酶間的結合。這種假設在哺乳動物和酵母 FKBP12上得到了驗證, 二者分別與脯氨酸類似物FK506和雷帕霉素結合后, 抑制CaN激酶活性和 TOR磷酸酶活性[6]。Geisler等[30]發(fā)現TWD1可與PINOID激酶結合, 從而調控ABCB1生長素轉運蛋白的磷酸化, 另外哺乳動物 FKBP52促進 AKT激酶和 PHLLP磷酸激酶的結合[83], 說明FKBP可參與調控靶蛋白的磷酸化。但是目前還沒有證據表明FKBd或單結構域FKBP結合靶蛋白后可直接與激酶或磷酸酶結合。

5 結 語

植物FKBP在信號轉導、脅迫響應、光合作用以及基因轉錄等方面都具有重要作用。然而, 部分FKBP對植物的生理生化過程具有重要功能而其他卻功能冗余, 其 PPIase活性具有什么功能目前也未知, 因此植物FKBP的保守結構域FKBd的主要功能還不能確定。植物FKBP通過其FKBd結構域與特有的分子伴侶蛋白結合, 這些蛋白的多樣性和復雜性說明, FKBd通過進化發(fā)生了變異從而可與不同蛋白結合。最近幾年, 在植物 FKBP基因克隆與功能分析方面取得了一定進展, 但是植物 FKBP與其底物相互作用的機制, 還有待深入研究。為了更加清楚的了解該基因家族的全部特征, 需要通過基因工程, 獲得所有基因的轉基因株系, 進而系統(tǒng)研究該家族中各成員的功能。同時, 也可以通過獲得各個基因的突變體來研究基因的功能。相信隨著分子生物學技術的迅猛發(fā)展, 可以為將來植物 FKBP基因功能的深入研究提供一個有力的技術平臺。

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(責任編委: 陳受宜)

Structure and biological functions of plant FKBP family

Yanli Yu, Yanjiao Li, Kaiyuan Pang, Fajun Zhang, Qi Sun, Wencai Li, Zhaodong Meng

National Engineering Laboratory for Wheat and Maize, Institute of Maize, Shandong Academy of Agricultural Sciences, Jinan 250100, China

FK506-binding proteins (FKBPs) are well known as both the receptor for the immunosuppressant drug FK506 and the prolyl isomerase (PPIase) enzyme. FKBPs are widely and constitutively expressed, and highly conserved during evolution. In higher plants, FKBPs usually form a relative large and diverse family compared with that in other eukaryotes, and serve as important molecular chaperones that interact with specific protein partners to regulate a diversity of cellular processes which mainly influence the plant development and stress responding. More recently, studies discovered a series of new interacting partners of FKBPs, which implicate FKBPs in gene expression regulation and photosynthetic adaptation. This review mainly focuses on the structural characteristics, classification, and the latest discoveries in the physiological functions of FKBPs in higher plants.

FKBP; structural characteristics; biological functions; immunophilin

2013-10-12;

2013-12-09

轉基因生物新品種培育科技重大專項(編號：2013ZX08003-001), 山東省自然科學基金項目(編號：ZR2013CQ036), 山東省現代農業(yè)產業(yè)技術體系建設經費(編號：SDAIT-01-022-04)和山東省農業(yè)生物資源創(chuàng)新利用專項資金資助

于彥麗, 博士, 副研究員, 研究方向：玉米分子育種與分子生物學。Tel: 0531-83179313; E-mail: lily_yu74@hotmail.com

孟昭東, 博士, 研究員, 研究方向：玉米遺傳育種。E-mail: mengzd@saas.ac.cn

10.3724/SP.J.1005.2014.0536

時間: 2014-4-25 14:13:39

URL: http://www.cnki.net/kcms/detail/11.1913.R.20140425.1413.002.html